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Activity-Based Profiling of Proteases BI83CH11-Bogyo ARI 3 May 2014 11:12 Activity-Based Profiling of Proteases Laura E. Sanman1 and Matthew Bogyo1,2,3 Departments of 1Chemical and Systems Biology, 2Microbiology and Immunology, and 3Pathology, Stanford University School of Medicine, Stanford, California 94305-5324; email: [email protected] Annu. Rev. Biochem. 2014. 83:249–73 Keywords The Annual Review of Biochemistry is online at biochem.annualreviews.org activity-based probes, proteomics, mass spectrometry, affinity handle, fluorescent imaging This article’s doi: 10.1146/annurev-biochem-060713-035352 Abstract Copyright c 2014 by Annual Reviews. All rights reserved Proteolytic enzymes are key signaling molecules in both normal physi- Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org ological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochemical methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the ap- plication of ABPs to protease enzymology, discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers. 249 BI83CH11-Bogyo ARI 3 May 2014 11:12 gens that contain inhibitory prodomains that Contents must be removed for the protease to become active. Proteases that are not synthesized with INTRODUCTION.................. 250 inhibitory prodomains often require cofactor STRATEGIES FOR PROFILING binding or posttranslational modification for PROTEASEACTIVITY.......... 250 activation. After activation, endogenous protein NaturalSubstrateTurnover........ 251 inhibitors, changes in subcellular localization, Substrate-Based Probes . 253 and degradation can limit protease activity Activity-BasedProbes.............. 254 (Figure 1). Given the high level of posttransla- DESIGNING ACTIVITY-BASED tional regulation, it is not surprising that mea- PROBESFORPROTEASES...... 255 sures of protease activity, rather than traditional Reactive Functional Groups . 255 transcriptomic or proteomic analyses, are often Recognition Sequence Design . 258 necessary to understand biological function ReporterTags..................... 259 (4). The purpose of this review is to highlight APPLICATIONS OF recent methods that allow dynamic measure- ACTIVITY-BASED PROFILING ment of protease activity in complex biological TOPROTEASES................. 261 systems. We emphasize the relatively new, but Protease Enzymology . 261 rapidly growing, field of activity-based pro- Drug Discovery and Development . 263 filing, which has proven valuable for detailed Profiling Proteases in Disease . 265 analyses of proteases and other enzyme classes. INTRODUCTION STRATEGIES FOR PROFILING The basic definition of a protease is an enzyme PROTEASE ACTIVITY that hydrolyzes peptide bonds in proteins. Ap- The most direct method of tracking protease proximately 700 enzymes, or roughly 2% of the activity is monitoring hydrolysis of a substrate. human genome, have known or putative prote- Turnover of both natural protein substrates olytic activity (1). Although all share the ability and substrate-based probes can provide a direct to catabolize proteins, these enzymes vary in measure of protease activation events (5). The almost every other parameter, including size, quantity of peptide fragments that are pro- localization, and quaternary structure. For sim- duced by substrate proteolysis reflects levels of plification, proteases are grouped into families protease activity, but these substrate fragments on the basis of catalytic mechanism. In humans, must be isolated from their native environ- there are five protease families: aspartyl, cys- ment for identification and quantification. In Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org teine, metallo-, serine, and threonine proteases. contrast, substrate-based probes are reagents Cysteine, serine, and threonine proteases use that are designed to directly generate a signal by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. nucleophilic active-site residues to hydrolyze upon cleavage (6). Protease activity can also peptide bonds, whereas aspartyl and metallo- be indirectly monitored using activity-based proteases use active-site residues to activate wa- probes (ABPs), which contain a substrate-like ter molecules for nucleophilic attack (2). region but, rather than being processed into Proteolytic activity is central to many fragments, covalently bind to the active pro- necessary biological processes, including tease in an enzyme-catalyzed reaction (6–8). development, differentiation, and the immune Thus, the extent of target protease modifica- response (3). However, due to the irreversible tion reports levels of activity. Each of these nature of proteolysis, proteolytic activity must strategies for determining protease activity has be highly regulated within cellular systems. been applied in diverse biological systems, and Most proteases are translated as inactive zymo- each has its own strengths and weaknesses. 250 Sanman · Bogyo BI83CH11-Bogyo ARI 3 May 2014 11:12 abc Protease inhibitor PTMs Inhibition Inactive zymogen Active protease Environment Proteolytic prodomain removal Degradation Figure 1 Proteases are often synthesized as inactive zymogens (left) that are activated by several mechanisms, including posttranslational modifications (PTMs), cofactor binding, pH change, and proteolytic prodomain removal (center). Prodomain hydrolysis ( yellow stars) can occur at a macromolecular signaling complex (top), by the action of another individual protease (middle), or by autocatalysis (bottom). After activation, protease activity can be limited by endogenous inhibitors or by degradation by the proteasome (right). Below, we discuss examples of applications and Through the use of this method, many proteins the relative merits of each strategy. can be simultaneously monitored. Gel-free enrichment methods for proteolytic peptides have also been improved, allowing for simul- Natural Substrate Turnover taneous identification and quantification of Active proteases cleave protein substrates. The the majority of substrate hydrolysis events in a most direct example of this process is autopro- given sample (5, 12). These enrichments capi- teolysis, during which a protease catalyzes its talize on the unique α-amino group exposed by own processing to become active (9, 10). The substrate proteolysis. Proteolytic peptides can simplest measure of turnover of individual sub- be enriched for by negative selection, as in the strates of interest uses sodium dodecyl sulfate COFRADIC (13) and TAILS (14) methods, or polyacrylamide gel electrophoresis (SDS- by positive selection, as with selective α-amine PAGE) to resolve proteins and fragments, fol- biotinylation by subtiligase (15) or selective lowed by Coomassie staining or immunoblot- lysine capping using O-methylisourea followed Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org ting for protein visualization. However, this by biotinylation of α-amines (16). procedure requires a priori knowledge of Increasing coverage of the proteolytic by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. the substrates to be measured. Alternatively, “peptidome” by use of gel-based and gel-free turnover of all protease substrates can be mea- enrichment methods has facilitated systems- sured simultaneously by mass spectrometry level monitoring of protease activity. The (Figure 2). For example, in a technique named biological pathways into which protein sub- PROTOMAP, a proteome sample is prepared strates fall suggest protease function, and before and after addition of a protease enzyme. common cleavage sequences indicate substrate The samples are then resolved by SDS-PAGE, recognition motifs. Both have been useful for and the locations of all proteins and protein separating the biological functions of closely fragments are mapped in the gel by mass spec- related proteases and advancing the design trometry. Changes in the migration of protein of selective substrate-based probes and ABPs. fragments indicate substrate proteolysis (11). The following examples illustrate how methods www.annualreviews.org • Activity-Based Profiling of Proteases 251 BI83CH11-Bogyo ARI 3 May 2014 11:12 abc R O R1 Substrate 3 H N OH SH C terminus N N H O H O R2 R O R1 O R Substrate 3 H H –2 H Substrate + C terminus N N N N N N N terminus O R–2 H OOR H R H O H 2 –1 H N Substrate 2 N N N terminus Full-length substrate R H O Intensity –1 m/z Cleavage products Readouts Figure 2 Cleavage of substrate proteins reports protease activation. (Left) A cysteine protease (gray; active-site sulfhydryl group represented by –SH) hydrolyzes an amide bond, releasing two polypeptides (middle). (Right) Potential measures of substrate truncation include (top right) immunoblotting or Coomassie stain as well as (bottom right) mass spectrometry. Abbreviation: m/z, mass-to-charge ratio. of assessing natural substrate
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